HIV entry inhibitors comprise a new class of antiretroviral drugs that inhibit one of several steps in the virus entry pathway, including CD4-induced conformational changes in gp120, coreceptor binding, and membrane fusion. One membrane fusion inhibitor has been licensed (T20/enfuvirtide), three CCR5 inhibitors will be tested in Phase III clinical trials in 2005, a new CXCR4 inhibitor will soon be used in HIV-infected patients, and several other entry inhibitors are in preclinical and early clinical development. Thus, it can be anticipated that this new class of antiretroviral drugs will assume increasingly greater clinical importance in the years to come. However, the use of entry inhibitors will likely be complicated by the highly variable and plastic nature of the HIV-1 envelope (Env) protein, which results in considerable variability in the sensitivity of virus strains to entry inhibitors, which may result in varied and complex drug resistance pathways some of which could, through alterations in Env, affect virus tropism and pathogenicity. In addition, poorly defined host cell factors have a significant impact on the potency of some entry inhibitors, at least in vitro. Therefore, the primary goals of this R01 proposal are to not only use entry inhibitors as tools to understand Env function, but to use our knowledge of the HIV entry process to better understand how virus and host factors affect entry inhibitor sensitivity, how HIV acquires resistance to entry inhibitors in vivo, and how resistance impacts Env function, sensitivity to other entry inhibitors and neutralizing antibodies. To do this, we will take advantage of a wealth of assays and reagents that we have developed over the past 9 years of this award, as well as collaborations with Dr. Steven Deeks (UCSF) and Joseph Eron (UNC), as the focus of this grant has evolved from studying how HIV enters cells, to how to stop it as effectively as possible. We therefore propose 5 Specific Aims: 1) Define mechanisms by which HIV acquires resistance to T20 in vivo;2) Define the nature of compensatory mechanisms;3) Study the effect of entry inhibitor selective pressure on viral tropism;4) Define the host cell factors that influence entry inhibitor sensitivity;and 5) Characterize resistance to R5 and X4 inhibitors in vitro and in vivo.